GNAO1 R209C mutation activates PI3K/Akt/mTOR signaling. (A) Western blot (WB) analysis of phosphorylation of Akt (p-Akt), STAT-3 (p-STAT3), Src (p-STAT3), and Erk1/2 (p-Erk1/2) in Reh cells with ectopic expression of Flag-tagged WT GNAO1, the R209C mutant, or an empty vector (EV) control. (B) WB of GNAO1, p-Akt, and p-Erk1/2 in the PBMC samples from twin B before onset, twin A at onset, and twin A at remission. (C) Effects of treatment with PI3K inhibitor LY294002 on p-Akt and p-S6K stimulation in Reh cells expressing WT GNAO1, the R209C mutant, or an EV control. Cells were treated with LY294002 (10 μM) for 1 hour. (D) Effects of treatment with the mTORC1 inhibitor rapamycin on p-Akt and p-S6K stimulation. Cells were treated with rapamycin (250 nM) for 1 hour. Data are representative of 3 independent experiments with similar results.